Heme Binding Mechanism of Structurally Similar Iron-Regulated

Nov 18, 2013 - Near transporter (NEAT) domains of the iron-regulated surface determinant ... the import of nutritional heme from host animals to Gram-...
1 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/biochemistry

Heme Binding Mechanism of Structurally Similar Iron-Regulated Surface Determinant Near Transporter Domains of Staphylococcus aureus Exhibiting Different Affinities for Heme Yoshitaka Moriwaki,*,† Tohru Terada,‡ Jose M. M. Caaveiro,§ Yousuke Takaoka,∥ Itaru Hamachi,∥,⊥ Kouhei Tsumoto,§,@,# and Kentaro Shimizu† †

Department of Biotechnology and ‡Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan § Institute of Medical Sciences, The University of Tokyo, Tokyo 108-8639, Japan ∥ Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Nishikyo-Ku, Kyoto 615-8510, Japan ⊥ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan @ Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku 113-8656, Japan # Department of Medical Genome Science, School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8562, Japan S Supporting Information *

ABSTRACT: Near transporter (NEAT) domains of the ironregulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Grampositive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7−β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

A

The Isd proteins IsdH, IsdB, IsdA, and IsdC are anchored to the cell wall and display one or more copies of the Near transporter (NEAT) domain. IsdH-NEAT1 and -NEAT2 domains17−19 and the IsdB-NEAT120 domain bind to hemoglobin (or haptoglobin−hemoglobin). IsdH-NEAT3 (IsdH-N3), IsdB-NEAT2 (IsdB-N2), IsdA-NEAT (IsdA-N), and IsdC-NEAT (IsdC-N) domains capture free heme extracted from hemoglobin and transfer it to downstream proteins. It is important to note that there are two main types

cquisition of iron nutrients is essential for survival and proliferation of bacterial pathogens.1−4 Although most mammalian host organisms, including humans, also have abundant iron, the amount of free iron is very limited because it has low solubility and is often chelated by heme and/or proteins (i.e., lactoferrin).5,6 However, recent studies have revealed that Staphylococcus aureus, Bacillus anthracis, and other Gram-positive pathogens7−9 have evolved the iron-regulated surface determinant (Isd) system to steal iron from the heme group of hemoglobin.10−13 The Isd system is activated in response to iron depletion in the bacteria10 and is composed of several proteins that extract, transport, and metabolize heme.10,14−16 © 2013 American Chemical Society

Received: June 27, 2013 Revised: November 17, 2013 Published: November 18, 2013 8866

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

Article

Figure 1. (A) Sequence alignments of heme-binding NEAT domains in S. aureus. All the regions of NEAT domains were annotated in UniProt. The alignment was generated using Jalview version 2.831 and the T-Coffee algorithm.32 A black circle indicates the loop 1 region. (B−E) Arrangement of residues around the heme-binding pocket of IsdH-N3 (PDB entry 2Z6F), IsdB-N2 (PDB entry 3RUR), IsdA-N (PDB entry 2ITF), and IsdC-N (PDB entry 2O6P), respectively.

difference in affinity. The atomistic mechanism of heme transfer therefore remains unclear. In this study, we used experimental and theoretical approaches to explore the dominant factor determining the direction of heme transfer between the NEAT domains. We also used non-iron metalloporphyrins to examine the role of metal ions in the transfer. We measured free energy changes upon binding of heme for IsdH-N3, IsdA-N, and IsdC-N and compared the values with theoretical calculations obtained by molecular dynamics (MD) simulations. We identified important residues for the efficient transfer of heme based on the results of these MD simulations. The theoretical predictions were verified experimentally using mutants and a chimera of the NEAT domains.

of NEAT domains: hemoglobin-binding and heme-transferring. Although they share a similar three-dimensional structure and are linked to each other in IsdH and IsdB, the first type binds hemoglobin and the second type binds heme.19 For example, a recent study has shown that the IsdH-N2-N3 bidomain unit efficiently extracts heme from hemoglobin.18 Given that the heme molecule thus extracted binds to the N3 part of the bidomain, the heme-transfer relay starts with IsdH-N3 (or IsdB-N2). IsdE, which is anchored in the plasma membrane along with IsdF, receives heme from IsdC and imports it into the cytoplasm.21,22 Finally, IsdG and IsdI degrade heme to produce iron.23−25 IsdH and IsdB are located in the outermost region of the cell wall, whereas IsdA and IsdC are located in the middle and innermost regions of the cell wall, respectively. Heme transfer occurs effectively from IsdH-N3 to IsdA-N to IsdC-N or, alternatively, from IsdB-N2 to IsdA-N to IsdC-N.26 Heme is transferred rapidly from upstream to downstream NEAT domains and between identical NEAT domains.27−29 The efficient transfer of heme through the thick cell wall (20−80 nm in Gram-positive bacteria30) may be optimized by the sequential arrangement of the Isd proteins in the cell wall and by the rapid transfer of heme between NEAT domains. The tertiary structures of the NEAT domains have revealed unique interactions between the NEAT domains and heme. In all the NEAT domains, the ferric iron atom of heme is coordinated by a conserved tyrosine residue, which is uncommon in most heme-binding proteins, in which a histidine residue coordinates iron (Figure 1).33−36 In addition, a propionate group of heme forms hydrogen bonds with some of the surrounding residues (Figure 1). As downstream NEAT domains have a higher affinity for heme, an affinity-driven heme-transfer mechanism has been proposed.34 However, as the structures of the NEAT domains are quite similar to each other, it is difficult to explain the physicochemical basis of the

■

MATERIALS AND METHODS Preparation of Metalloporphyrins and Isd Proteins. Hemin and Mn(III)-PPIX chloride were purchased from Frontier Scientific (Logan, UT). Ga(III)-PPIX hydroxide was synthesized according to the methodology of Nakae et al.37 As reported previously,38 cloning, expression, and purification of heme-free (apo) proteins IsdH-N3 (comprising residues Gly-534−Gln-664) and full-length IsdA except for the N- and C-terminal signal sequences (residues Ala-47−Thr-316) were performed using Escherichia coli RP523,39 a hemB mutant strain. IsdA-N (residues Ser-62−Ala-184) and full-length IsdC except for the N- and C-terminal signal sequences (residues Ala-28− Thr-192) were amplified by polymerase chain reaction and cloned into a pTAC-MAT-Tag-2 expression vector (SigmaAldrich, St. Louis, MO). Expression and purification were conducted as described above. The His6 tag was always cleaved off with thrombin protease (Sigma-Aldrich). Isothermal Titration Calorimetry (ITC) Analysis. Thermodynamic parameters of the interaction between the NEAT domains and metalloporphyrins were determined with 8867

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

Article

iTC 200 (GE Healthcare) at 25 °C. The apo form of IsdH-N3, IsdA-N, full-length IsdC, or full-length IsdC mutants were equilibrated with buffer [0.2 M NaCl and 50 mM Tris-HCl (pH 8.0)]. It is important to note that these protein samples did not contain heme derived from the expression system. Proteins filled in the calorimeter cell were diluted to a concentration of 20 μM with buffer containing 5% (v/v) dimethyl sulfoxide (DMSO). A solution of each metalloporphyrin with 5% (v/v) DMSO (375 μM) was prepared from powder for each experiment and filled into the calorimeter syringe. We also measured the affinity of IsdH-NEAT3 at 15, 20, and 30 °C to evaluate its temperature dependence. The binding isotherms were fit to a one-site binding model using ORIGIN version 7.0. Heme Transfer between IsdH-N3 and IsdC Measured by UV−Visible Absorbance Spectroscopy. The transport of heme from IsdH-N3 to IsdC was measured according to the methodologies of Liu et al.27 The holo form of IsdH-N3 (14 μM) and the apo form of IsdC (16 μM) were mixed in 1 mL of 50 mM sodium phosphate buffer (pH 7.3) for 1 or 2 min. The proteins were subsequently separated by ion-exchange chromatography using a diethylaminoethyl Sepharose (DEAESepharose) column. IsdH-N3 binds to the column, whereas full-length IsdC appears in the flow-through eluate. IsdH-N3 was recovered with a solution containing 500 mM NaCl. To examine the transfer of metalloporphyrins, we measured the intensities of the Soret band [heme, 403 nm; Ga(III)-PPIX, 413 nm; Mn(III)-PPIX, 375 nm] for both IsdH-N3 and IsdC using a Jasco (Tokyo, Japan) spectrophotometer. Construction of Initial Structures of MD Simulations. Crystal structures have been determined for heme-bound (holo) forms of IsdH-N3 (PDB entry 2Z6F), IsdA-N (PDB entry 2ITF), and IsdC-N (PDB entry 2O6P), and for hemefree (apo) forms of IsdH-N3 (PDB entry 2E7D) and IsdA-N (PDB entry 2ITE). Their coordinates were downloaded from the PDB and used as the initial structures of the MD simulations. As the crystal structure of the apo form of IsdC-N has not been determined, the structure of its apo form was constructed by deleting heme from its holo structure. The structure of a loop 1 chimera between IsdC-N and IsdH-N3 (IsdC-N-loop 1H) was modeled by superposing the Cα atoms of the holo structure of IsdC-N upon those of the holo structure of IsdH-N3 and by subsequently substituting the loop 1 region (residues Tyr-39−Thr-46) of IsdC-N for the corresponding region (Phe-555−Glu-562) of IsdH-N3 (Table S1 of the Supporting Information). The structure of the IsdCN-F130A mutant was constructed using the “mutagenesis” function implemented in PyMOL.40 The N- and C-termini of all the protein models were capped with an acetyl group and an N-methyl group, respectively. The protonation states of the histidine residues at pH 7.0 were determined by PROPKA3.41 The deprotonated form was used for the tyrosine residues coordinating to the iron of heme (Y642 of IsdH-N3, Y166 of IsdA-N, and Y132 of IsdC-N), not only in the MD simulations of the holo forms but also for the apo forms because the MD simulation with the protonated form performed for the apo form of IsdA-N did not reproduce the hydrogen bond between Y166 and Y170 observed in its crystal structure. Water molecules and charge-neutralizing ions were placed around each protein using Solvate version 1.0.42 Water molecules were further added to the system with a thickness of more than 5 Å to form a box-shaped system using the LEaP module of AmberTools version 1.5.43 The Amber ff99SB force field44 was

employed for the proteins, and the TIP3P model45 was used for the water molecules. The partial charges of deprotonated tyrosine and heme bound to deprotonated tyrosine or chloride ions were determined as follows. We first optimized the structures of the 4-methylphenoxide, heme 4-methylphenoxide, and heme chloride at the B3LYP/6-31G(d)46,47 level using Gaussian 09.48 The electrostatic potentials around the molecules were then calculated. The partial charges were determined to fit the electrostatic potentials using the restrained electrostatic potential (RESP) method,49 implemented in the ANTECHAMBER module of AmberTools version 1.5. It is important to note that we used two different sets of partial charges for the apo and holo form simulations. Bond, angle, and torsion parameters describing the interaction between heme and deprotonated tyrosine were determined on the basis of the energy changes caused by small structural changes. The partial charges and parameters used here are described previously.50 Each MD simulation was performed as follows. First, the positions of the water molecules and the counterions were optimized by a 200-step energy minimization. The whole system was then subjected to a 200-step energy minimization. The system was heated gradually to 300 K in a 200 ps constantNVT MD simulation with harmonic position restraints imposed on all heavy atoms of the solutes with a force constant of 10 kcal mol−1 Å−2. During the subsequent 800 ps constant-NPT simulation, the restraints were gradually relaxed and the system was equilibrated at 300 K and 1.0 × 105 Pa. The SHAKE algorithm51 was employed to constrain all bond lengths involving hydrogen atoms, which allowed the use of a time step of 2 fs. Electrostatic interactions were calculated using the particle mesh Ewald (PME) method,52,53 with a cutoff radius of 8 Å. The temperature was controlled by the Langevin dynamics algorithm54 with a collision frequency of 2.0 ps−1. The pressure was controlled using the weak coupling method.55 The minimization and equilibration runs were conducted using the Sander or PMEMD module of AMBER 11.43 A production run was conducted for 200 ns, maintaining the temperature at 300 K and the pressure at 1.0 × 105 Pa with GROMACS version 4.5.5.56 The AMBER topology files were converted into the GROMACS format using the ffAmber script.57 During the production run, the P-LINCS algorithm58 was employed to constrain the bond lengths involving hydrogen atoms. Calculation of Binding Free Energy using the MM− PB/SA Method. The molecular mechanics−Poisson−Boltzmann/surface area (MM−PB/SA)59 method was employed to calculate the binding free energy, ΔGbind, for the complexes between the NEAT domains and heme. Here, we considered the following reaction: protein + heme‐Cl → protein−heme + Cl−

where protein is IsdH-N3, IsdA-N, or IsdC-N and the tyrosine residue to which the heme iron binds is deprotonated in both the apo and holo forms. In the MM−PB/SA method, ΔGbind was calculated as 8868

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

Article

Table 1. Results of the ITC Experiment with Isd Proteins and Metalloporphyrinsa metalloporphyrin heme

Mn-PPIX

Ga-PPIX

a

ΔH° (kcal/mol)

protein

−10.6 −11.0 −14.6 −5.77 −9.5 −14.6 −7.67 −8.0 −13.2

IsdH-N3 IsdA-N IsdC IsdH-N3 IsdA-N IsdC IsdH-N3 IsdA-N IsdC

± ± ± ± ± ± ± ± ±

−TΔS° (kcal/mol)

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.39 0.30 3.4 −2.84 −1.8 3.25 −1.97 −3.0 0.95

ΔG° (kcal/mol) −10.2 −10.8 −11.2 −8.61 −11.3 −11.4 −9.62 −11.0 −12.2

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.2

Kd (nM) 34 14 6.5 490 5.7 4.5 91 34 14

± ± ± ± ± ± ± ± ±

8 4 1.4 60 2.1 1.0 21 8 4

Temperature of 25 °C (298.15 K).

Principal Component Analysis. Principal component analysis (PCA) was performed for the MD trajectories of the apo and holo forms of the wild-type IsdC-N protein and the IsdC-N-F130A mutant. The variance−covariance matrix was calculated from the Cα atom coordinates of the MD trajectories between 10 and 200 ns after superimposing them onto their average coordinates. The matrix was then diagonalized to obtain the principal component eigenvectors. Every structure in the MD trajectories was projected onto the space defined by the first and second principal component eigenvectors to graphically display the conformational distribution. Cloning, Expression, Purification, and Binding Analysis of the Isd Mutants. To verify the predictions by the MD simulations, four mutants of full-length IsdC were prepared with a Takara Bio mutagenesis kit. A full-length IsdC-E88A mutant, the full-length IsdC-loop 1H chimera, and a full-length IsdC-F130A mutant were constructed using the pairs of primers described in Table S2 of the Supporting Information. A double mutant of the full-length loop 1H chimera and E88A (IsdC-E88A-loop 1H) was also constructed by combining the preceding primer sets. As a complementary approach, we also constructed an IsdH-N3-loop 1C chimera, whose loop 1 region (residues Phe-555−Glu-562) is substituted with the corresponding region (Tyr-39−Thr-46) of IsdC (Table S1 of the Supporting Information). Expression and purification were performed as described above. The binding affinities of these mutants for heme were determined by ITC in the same manner described above. The concentration of full-length IsdC-E88A-loop 1H in the calorimeter cell was 12 μM. Heme Transfer between Three Isd Mutants and IsdA. As IsdA (also IsdA-N) and the IsdC mutants were not well separated by ion-exchange chromatography, we used a sizeexclusion chromatography column, HiLoad 16/60 Superdex 200 (GE Healthcare), to separate these two proteins (Figure S1 of the Supporting Information). Note that full-length IsdA was eluted from the column at the volume equivalent to that of a 100.3 kDa protein.64 The holo form of full-length IsdC-F130A (16 μM) and the apo form of full-length IsdA (16 μM) were mixed in a 3.5 mL solution and then injected into the column and equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl. Analyses of two heme transfers between the holo form of full-length IsdC-E88A-loop 1H and the apo form of full-length IsdA and between the holo form of IsdH-N3-loop 1C and the apo form of full-length IsdA were performed in a similar manner.

ΔG bind = EMM(protein−heme) − TS(protein−heme) −

−

+ ΔGsolv (protein−heme) + EMM(Cl ) − TS(Cl ) + ΔGsolv (Cl−) − [EMM(protein) − TS(protein) + ΔGsolv (protein) + EMM(heme‐Cl) − TS(heme‐Cl) + ΔGsolv (heme‐Cl)] + ΔEcorr

where EMM(X) and S(X) are molecular mechanical energy and entropy of X calculated in the gas phase, respectively, T is the temperature (298.15 K), ΔGsolv(X) is the solvation free energy, and ΔEcorr is a correction term. EMM(X) was calculated as the sum of the bond, angle, torsion, van der Waals, and electrostatic energies. The vibrational component of S(X) was estimated by the quasi-harmonic approximation method.60,61 ΔGsolv(X) was calculated as the sum of the polar [ΔGpol(X)] and nonpolar [ΔGnp(X)] contributions, except for ΔGsolv(Cl−), for which the experimental value (−74.7 kcal mol−1)62 was used. ΔGpol(X) was calculated by solving the Poisson−Boltzmann equation with interior and exterior dielectric constants of 1 and 80, respectively, with a salt concentration of 0.2 M. ΔGnp(X) was approximated using a linear function of the solvent accessible surface area (SASA) of solute X as ΔGnp(X) = γ SASA + b

where γ and b were 0.00542 kcal mol−1 Å−2 and 1.008 kcal mol−1, respectively. The solvent probe radius used in the calculation of SASA was set at 1.4 Å. The correction term, ΔEcorr, was added to consider the energy change due to the cleavage and formation of the chemical bonds, which was calculated approximately as ΔEcorr = EQM(4MP−heme) + EQM(Cl−) − [EQM(4MP−) + EQM(heme‐Cl)] − {EMM(4MP−heme) + EMM(Cl−) − [EMM(4MP−) + EMM(heme‐Cl)]}

where EQM(X) is the quantum mechanical (QM) energy of X calculated at the B3LYP/6-31G(d) level and 4MP stands for 4methylphenoxide. In addition, we also calculated the polar contributions to ΔGsolv(protein−heme), ΔGsolv(protein), and ΔGsolv(heme-Cl) using a generalized Born (GB) model to decompose the energy changes into per-residue contributions.63 All the calculations were performed using the MD trajectories between 10 and 200 ns recorded every 20 ps (9500 snapshots for each protein). 8869

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

■

Article

Direct Transfer of Metalloporphyrin from IsdH-N3 to IsdC. Direct transfer of heme from IsdH-N3 to IsdC has been demonstrated by electrospray ionization mass spectrometry (ESI-MS).26 In this study, we determined the direct transfer of metalloporphyrins from IsdH-N3 to IsdC using UV−visible spectroscopy. After incubation of holo-IsdH-N3 with full-length apo-IsdC for 2 min and subsequent separation on a DEAE ionexchange column, the solution containing IsdH-N3 showed a marked decrease in the intensity of the Soret band. In contrast, the intensity of the Soret band of the IsdC sample increased markedly (Figure 2 and Figure S3 of the Supporting

RESULTS AND DISCUSSION Binding of Heme and Metalloporphyrins to NEAT Proteins. The thermodynamic parameters of the binding of heme or metalloporphyrins to the NEAT domains were determined by ITC (Table 1). The injection of heme into protein solutions containing NEAT domains released a large amount of heat. As shown in Table 1, the magnitude of exothermic heat increased from IsdH-N3 (−10.6 ± 0.1 kcal mol−1) to IsdA-N (−11.0 ± 0.1 kcal mol−1) to IsdC (−14.6 ± 0.1 kcal mol−1). The dissociation constant of IsdC was consistently the smallest (Kd = 6.5 ± 1.4 nM), and therefore, it exhibited the strongest affinity for heme. The Kd of IsdA-N was intermediate (14 ± 4 nM), and that of IsdH-N3 was the largest (34 ± 8 nM). The order of affinities corresponded to the reported biological order of heme transport across the cell wall mediated by the NEAT domains. Interestingly, the difference in ΔH° between IsdA-N and IsdC was 3.6 kcal mol−1, whereas that for ΔG° was only 0.4 kcal mol−1, indicating a relatively larger entropy loss associated with the binding of heme to IsdC-N. This observation was consistent with a previous report that showed the hemebinding pocket of IsdC-N is flexible in the apo form and becomes rigid upon binding to heme.65 The affinity of heme was 2−3-fold larger for IsdC than for IsdA. This value is smaller than that determined in a previous kinetic report (5−15fold).27 The discrepancy may be due to differences in experimental conditions (protein concentration or methods) and the binding by heme as a dimer in our ITC experiments. However, we note that the trend of affinities is consistent with the previous report. We next measured the Kd values of Mn-PPIX and Ga-PPIX, two closely related analogues of heme.38,66 The results of ITC indicated that all the metalloporphyrins tested bound to each of the three NEAT domains with similar affinities. Furthermore, the Kd values for Mn-PPIX and Ga-PPIX became smaller for the downstream NEAT domains, similar to the Kd for heme (Table 1). However, the differences in ΔG° and ΔH° related to the change in metal ion were not constant between the NEAT domains. Notably, the affinities of Mn-PPIX and Ga-PPIX were both lower than that of heme in the case of IsdH-N3 but higher in the cases of IsdA-N and IsdC. Although the ITC data were collected at 25 °C, we expect that the order of the affinities among the NEAT domains is not changed at 37 °C. To evaluate the temperature dependence of the affinity, we measured the affinity of IsdH-NEAT3 for heme in the temperature range from 15 to 30 °C. Although the affinity could not be measured at 37 °C because of the instability of the baseline of the ITC instrument, the Kd value remained almost constant, within experimental error, in this temperature range (Table S3 of the Supporting Information). The stoichiometry indicated that two molecules of heme bind to IsdA-N and IsdC-N (Figure S2 of the Supporting Information), as previously explained for IsdH-N3.50 The same stoichiometry was obtained from experiments using non-iron metalloporphyrins. Given that heme forms dimers in the highconcentration solutions employed for the ITC experiments, we believe that heme and the non-iron metalloporphyrins bind to the NEAT domains as dimers under the conditions used for the ITC experiment. The thermodynamic parameters obtained from the ITC measurements are affected little by the binding of the extra heme molecule because the second heme molecule binds very weakly to the heme−NEAT domain complex.50

Figure 2. Transfer of heme between IsdH-N3 and IsdC. (A and C) IsdH-N3 spectra after elution from the DEAE column. (B and D) IsdC spectra. Heme bound to IsdH-N3 was effectively transferred to apoIsdC (A and B), whereas heme bound to IsdC was not effectively transferred to apo-IsdH-N3 (C and D). The transfer of Mn-PPIX and Ga-PPX is shown in Figure S3 of the Supporting Information.

Information). These changes in the spectra closely resembled those observed in our previous experiments using IsdH-N3 and full-length IsdA,38 indicating that metalloporphyrins were transferred effectively from IsdH-N3 to IsdC in the absence of the intermediary transporter IsdA-N. This rapid transfer also suggests that IsdH-N3 and IsdC form a heme-transfer intermediate complex like the IsdA−IsdC complex.28 Meanwhile, back-transfer of metalloporphyrins from full-length holoIsdC to apo-IsdH-N3 did not occur even after incubation for 20 min. These results suggest that the transfer of metalloporphyrin is controlled by thermodynamic equilibrium. Theoretical Values of Binding Free Energies. We estimated the free energies of binding of heme to IsdH-N3, IsdA-N, and IsdC-N using the MM−PB/SA method. The correction term, ΔEcorr, was calculated to be −104.5 ± 2.0 kcal mol−1. Calculated ΔGbind values and their components are listed in Table 2. The sufficiency of conformation sampling was assessed by monitoring the time evolution of the first principal component in the form of the slowest mode of variation for each of the apo and holo forms of the three NEAT domains (Figure S4 of the Supporting Information). We found that the 200 ns MD simulations were sufficient to obtain convergent results for all except the apo form of IsdH-N3, where a 8870

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

Article

Table 2. Binding Free Energy and Energy Decomposition of ΔG Calculated by the MM−PB/SA Method (kilocalories per mole)a contribution

IsdH-N3

IsdA-N

IsdC-N

IsdC-N-loop 1H

ΔEEEL ΔEvdw ΔEinternalb ΔGPB ΔGSA ΔGMMPBSAc −TΔSsolute ΔEcorrd ΔG

172.08 −37.72 −2.53 −85.37 −3.64 42.82 39.41 −104.5 −22.3

−219.64 −23.19 −2.82 267.90 −4.41 17.84 39.46 −104.5 −47.2

−133.83 −43.51 −7.05 197.49 −3.94 9.16 41.69 −104.5 −53.6

26.95 −39.84 1.90 40.04 −3.99 25.06 32.73 −104.5 −46.7

a

These data were calculated from 9500 snapshots during a 200 ns production run. bThe ΔEinternal term is a summation of ΔEbond, ΔEangle, and ΔEtorsion. cΔGMMPBSA term is a summation of ΔEEEL, ΔEvdw, ΔEinternal, ΔGPB, and ΔGSA. dΔEcorr is a term to correct the MM energy between heme-binding Tyr and the iron in heme to QM energy.

conformation change was observed at 150 ns. Although the ΔGbind values were overestimated compared with the ITC values, the order of affinity was completely consistent between these two techniques. ΔGbind of IsdC-N was the smallest, and ΔGbind of IsdH-N3 was the largest. Comparison of the free energy components revealed differences in the interaction mechanism between the proteins. IsdH-N3 showed a positive average electrostatic energy change (ΔEelec = 172.08 kcal mol−1), whereas IsdA-N and IsdC-N showed negative values (−219.64 and −133.83 kcal mol−1, respectively). This indicated that the electrostatic interaction between IsdH-N3 and heme was unfavorable (Table 2). Meanwhile, the change in the polar contribution to the solvation free energy, ΔGpol, was negative (−85.37 kcal mol−1) in IsdH-N3 and positive in IsdA-N and IsdC-N (267.90 and 197.49 kcal mol−1, respectively). These results indicate that favorable electrostatic interactions are formed between IsdA-N or IsdC-N and heme, accompanying the desolvation of interface residues, whereas the electrostatic interactions between IsdH-N3 and heme are not very favorable. To identify key residues for heme binding, ΔGbind was decomposed into the contribution of each residue. We used the GB model instead of the PB method to calculate the polar contribution of the solvation free energies. The contributions of backbone and side chain atoms of each residue to ΔGbind are plotted against residue number in Figure 3. On the basis of the comparison of the cumulative plots of these contributions (Figure 3E), we selected two interesting regions for further analysis that showed large differences in the values of ΔGbind among the Isd proteins. The first region was located near loop 1 between strand β1b and the 310 helix (Figure 3A). In this region, IsdH-N3 displayed several residues with unfavorable contributions to the binding of heme, particularly Glu-556 and Glu-559 (Figure 3B), whereas IsdA-N and IsdC-N have fewer unfavorable residues (Figure 3C,D). As a result, the loop 1 region of IsdH-N3 was less suitable for binding heme than IsdA and IsdC. Although the primary sequence of this region varies among the NEAT domains as shown in their sequence alignment (Figure 1A), the serine residue at the end of loop 1 region is well-conserved. This serine residue forms a hydrogen bond with the propionate group of heme in the crystal structures of all the NEAT domains. However, MD simulations suggested that the strength

Figure 3. Per-residue binding free energy decomposition. (A) Superposition of structures of IsdH-N3 (blue), IsdA-N (yellow), and IsdC-N (red). (B−D) Energetic contributions of each residue in IsdHN3, IsdA-N, and IsdC-N to the binding of heme, respectively. (E) Additive energies of each residue from the N-terminus. The loop 1 region is indicated by dashed lines in each panel.

of this hydrogen bond varied between the receptors. The plots of the time evolution of the hydrogen bond distances indicated that the hydrogen bond in IsdH-N3 was less stable than those in IsdA-N and IsdC-N (Figure 4A). Importantly, when the loop 1 region (residues 39−46) of IsdC-N was substituted with the corresponding region (residues 555−562) of IsdH-N3 (the IsdC-N-loop 1H chimera), the hydrogen bond became unstable like that of IsdH-N3 (Figure 4B). Consistent with this observation, the MM−PB/SA calculation for this chimera predicted that this substitution significantly reduced its affinity 8871

dx.doi.org/10.1021/bi4008325 | Biochemistry 2013, 52, 8866−8877

Biochemistry

Article

by ITC. ΔH° of the chimeric protein was 1.9 kcal mol−1 weaker than that of the wild type (Table 3). This observation was consistent with the prediction made from the MD simulation. The affinity was also weaker for the chimeric construct (ΔΔG° = 0.7 kcal mol−1). Next, we examined the effects of the E88A mutation of wild-type IsdC and the combined mutation with the IsdC-loop 1H construct using ITC. In agreement with the prediction of the MD simulation, we found that the E88A mutations increased ΔH° by 3.4 kcal mol−1 (from −14.6 to −11.2 kcal mol−1) and 3.6 kcal mol−1 (from −12.7 to −9.1 kcal mol−1) for the wild-type and chimera proteins, respectively (Table 3). In addition, this result indicated that the energetic effect of loop 1 and E88 on enthalpy was additive within acceptable margins of error. However, the increases in free energy were small for all the chimeric and mutant proteins [ΔΔG° = 0.7−0.9 kcal mol−1 (Table 3)]. This was probably due to the enthalpy−entropy compensation effect.67,68 When the interaction was not very favorable, the amino acids or the functional groups of the ligand in the binding interface remained flexible, which reduced the magnitude of the entropy loss upon binding. We measured the affinity of IsdH-N3-loop 1C in the same manner to determine whether the effect of the loop 1 region is reversible. The ΔH° and ΔG° values of this protein decreased by 0.7 and 1.1 kcal mol−1, respectively, from those of the wild type (Table 3). The results for IsdC-loop 1H and IsdH-loop 1C clearly indicate that the affinity of the Isd-NEAT domains for heme (and other metalloporphyrins) is influenced by the loop region. Role of F130 of IsdC. Phenylalanine 130 (F130) of IsdC is in the proximity of Y132, the tyrosine residue that is coordinated to the iron atom of heme. Sequence alignment indicates that this phenylalanine is unique to IsdC. Other NEAT domains have an isoleucine residue at the corresponding position (Figure 1A). We constructed a model of the F130I mutant of IsdC-N (IsdC-N-F130I) and performed MD simulations to investigate the role of this residue in heme binding. Comparison of the MD trajectories between the wild-type and mutant IsdC-N proteins indicates that the F130I mutation destabilized the hydrogen bond formed between the Oη atoms of Y132 and Y136 in the apo form (Figure 5). The distance between the two atoms was